Separation of magnetic particles from a fluid has been known as magnetic separation or high gradient magnetic separation (HGMS) for about 40 years. In magnetic separation, particles of larger (d.gtoreq.0.5 micron) are captured or separated and in HGMS, smaller particles are separated, for example colloidal magnetic particles. Magnetic particles are today widely available commercially, typically 1 micron in diameter, with or without functional groups capable of binding antibodies or DNA molecules or containing other binding sites for sample purification. Several commercial systems automate sample purification and detection using magnetic particles, the systems ranging in size from desk-top to bench size.
Over the past decade, sub-millimeter-scale, automated flow-based analyzers and chemical detector arrays have steadily approached the technology level needed for commercialization. Development is continuing toward ever more compact (briefcase size) medical diagnostic analyzers for automated immunoassays, DNA purification and amplification, cell separation, etc. Despite the advances in miniaturization, particle handling has remained somewhat unchanged.
Automation has been primarily with robotic imitation of manual procedures for handling the magnetic particles (Immunoassay Automation, Editor D. W. Chan, 1996, Academic Press) These systems include capture of the magnetic particles by placing the magnetic particle suspension in a container that is located in a magnetic field gradient (e.g. above a magnet), so that the magnetic particles settle and are held at the bottom of the container.
Baxter Biotech Immunotherapy has a system that includes stationary capture followed by capture during continuous flow. Their system includes collection of most of the magnetic particles in a stationary reservoir above a magnet, followed by flow of the remaining solution over another magnet to remove any magnetic particles that were not captured in the first stage (Cell Separation Methods and Applications, E. Recktenwald, A. Radbruch, Eds., 1998, Marcel Dekker, pg 193). All of these systems include particle capture only at the walls of the reservoirs or tubing, and the vast majority of the magnetic particles are held within one container while solution is decanted and added.
Pollema and Ruzicka (C. H. Pollema, J. Ruzicka, G. D. Christian, and A Lernmark, Analytical Chemistry, volume 64, pages 1356-1361, 1992) describe a method for handling magnetic particles in a flow system, however, their system includes particle capture only at the tubing walls, and therefore does not allow for efficient perfusion of captured particles. Similarly, R. Kindervater, W. Kunneke, and R. D. Schmid (Analytical Chimica Acta, volume 234, pages 113-117,1990) describe a magnetic capture device consisting of tubing in close proximity to a magnet as part of a flow system. S. Sole, S. Alegret, F. Sespedes, E. Fabregas, and T. Diez-Caballero describe a flow system using magnetic capture of beads at a planar sensor surface, using a magnet external to the flow path. This geometry does not provide efficient perfusion through a bed of magnetic particles.
Separations of colloidal superparamagnetic particles (20 nm to 100 nm in size) are done using high gradient magnetic fields in an apparatus as shown in FIG. 1. Magnetic particles 100 in a fluid 102 flow through a magnetic flux conductor 104 that is permeable. These are generally contained in a column 106 and a controllable magnet 108 external to the column 106 is used proximate the magnetic flux conductor 104 for adjusting the magnetic field within the magnetic flux conductor.
The flux conductor 104 was magnetic grade stainless steel wool 110 in U.S. Pat. Nos. 3,567,026 and 3,676,337 (1971). In U.S. Pat. No. 4,247,389 (1981), the stainless steel of the steel wool 110 was replaced with an amorphous metal alloy containing iron and cobalt.
Because bare metal contributed to oxidation of biological species, U.S. Pat. No. 4,375,407 (1983) presented a polymer coated steel wool (not shown) or filamentary magnetic material. Additional U.S. Pat. Nos. (5,385,707,1995; 5,411,863, 1995; 5,543,289,1996; 5,693,539,1997) rely on the use of polymer coated filamentary magnetic material alone or in combination with functionalized beads.
For capture of blood cells, U.S. Pat. No. 4,664,796 (1987) discusses magnetic spheres in combination with filamentary magnetic material.
Alternative forms of flux conductor 104 are discussed in U.S. Pat. Nos. 520,000,084,1993; 5,541,072, 1996; 5,622,831,1997; 5,698,271,1997. Specifically discussed are wire loops and arrays of thin rods.
An automated separation system that includes a HGMS column is available from Miltenyi-Biotec/AmCell. They use a peristaltic pump to pull samples through a ferromagnetic column. The column is used to capture cells that are pre-labeled with very small colloidal superparamagetic particles (20-100 nm in diameter) rather than larger superparamagnetic particles used for most applications (0.5-5 .mu.m in diameter). The Miltenyi-Biotec/Amcell columns contain a closely packed bed of ferromagnetic spheres coated with biocompatible polymer. The cells that are labeled with colloidal superparamagetic particles are captured at the surfaces of the spheres within the flow path. (Cell Separation Methods and Applications, E. Recktenwald, A. Radbruch, Eds., 1998, Marcel Dekker, pg 153-171)
The three dimensional structure and distribution of the magnetic flux conductor material influences fluid flow, magnetic field flux distributions, and hence particle capture efficiency, and the ability to uniformly perfuse the particles after capture. In addition, the structural geometry and magnetic field gradient define the range of particle sizes that can be efficiently captured and released. Columns packed with filamentary magnetic flux conductor material have a nonuniform distribution of the material resulting in variable magnetic flux distributions and nonuniform fluid flow. Reservoirs containing wire loops, rods or a piece of wire mesh have more uniform structure, but still have a non-uniform distribution of material in the reservoir, and previous work does not include perfusion of these structures in a column format (U.S. Pat. No. 5,200,084). Columns packed with spherical particles provide uniform magnetic flux distributions and uniform fluid flow, however the pressure drop across the column can be high since the porosity is low (only 20% porous if the spheres are uniform in size and not closely packed).
Heretofore, fluid permeable magnetic flux conductors suffer from one or more of the following disadvantages: non-uniform field gradient distributions, inefficient perfusion characteristics, or low porosity. First, the maximum distance from a particle to a flux conductor surface is not sufficiently small and uniform throughout the volume containing the flux conductor to promote efficient particle capture on the basis of distance to be traveled. Particles near the highest field gradient (e.g. regions of the flux conductor surface within the flow path) are captured while particles farther from the flux conductor are not captured unless the flow rate is reduced. Thus, particle capture is inefficient above a threshold flowrate that depends on the device dimensions and particle size. Non-uniform pore sizes can also lead to difficulty removing the particles if any pores are on the order of the particle size or smaller. The lack of uniformity also results in magnetic flux gradients unevenly distributed throughout the material. The present structures do not provide uniform fluid flow throughout the flow path. Therefore, particles are captured non-uniformly throughout the flow path (e.g. only at the non-uniformly distributed flux conductor surface, or regions of this surface) so that one cannot uniformly perfuse the captured particles. Some of the present structures also do not provide efficient perfusion of the flux conductor surface. [packed spheres do provide this, but suffer from low porosity and high pressure drop]. Thus, a particle traveling through the material does not necessarily come close to conductor material as it flows through the structure. An extreme example of this situation is flow through a tube of magnetic flux conducting material.
Finally, although a column of packed spheres provides the above advantages as long as the spheres are closely packed to prevent fluid channeling through large gaps, the packed bed has a low porosity (.about.20%) and therefore there is a high pressure drop across the magnetic flux material. In addition, the low porosity requires that the system size must be scaled up considerably to handle standard superparamagnetic particles (&gt;0.5 micron in size) rather than just colloidal superparamagnetic particles.
Another difficulty with the prior art methods is the inability to release 100% of the magnetic particles because of residual magnetism that remains in the magnetic flux conductor. Miltenyi (1997) 5,411,863 states:
"`Ferromagnetic` materials are strongly susceptible to magnetic fields and are capable of retaining magnetic properties when the field is removed . . . Ferromagnetic particles with permanent magnetization have considerable disadvantages for application to biological material separation since suspension of these particles easily aggregate due to their high magnetic attraction for each other." PA1 "A preferred embodiment shown in FIG. 1 utilized a permanent magnet to create the magnetic field . . . The magnet is constructed of a commercially available alloy of neodinium/iron/boron . . . Indeed, an electromagnet could be substituted in less preferred embodiments . . . If an electromagnet is used, the magnetic field created by the electromagnet is compensated to zero. Upon removal of the magnet field and continued flow of suspension fluid through the chamber, the retained magnetized particles are eluted from the matrix." PA1 a magnetic flux conductor that is permeable thereby permitting the magnetic particles and fluid to flow therethrough; and PA1 a controllable magnetic field for adjusting the magnetic field within the magnetic flux conductor for handling the magnetic particles. The present invention is an improvement wherein the magnetic flux conductor is a monolithic porous foam. PA1 (a) applying a magnetic field of a first polarity for retaining said magnetic particles in said magnetic flux conductor; and PA1 (b) reversing said magnetic field to an opposite polarity for releasing said magnetic particles from said magnetic flux conductor.
also, at the end of column 10 and beginning of column 11,
It is well known that compensating to zero does not eliminate residual magnetism. Thus, Miltenyi is not able to remove 100% of the magnetic particles from the matrix without high shear forces.
Thus, there is a need in the art of magnetic particle handling for an apparatus and method for magnetic particle handling that provides more uniform retention of particles and uniform flow perfusion of the retained particles, and more efficient removal of the particles for reuse of the system. The system should be suitable for handling magnetic particles ranging from about 100 nm to 10 .mu.m in diameter or magnetic colloids ranging from about 20 to 100 nm in diameter.